Embodiments of the present invention relate to spatially defined chemical synthesis involving lithographic processes. In particular, embodiments of the present invention are directed to novel methods and compositions for synthesizing arrays of diverse polymer sequences, such as polypeptides and polynucleotides. According to a specific aspect of the invention, a method of synthesizing diverse polymer sequences, such as peptides or polynucleotides, is provided. The diverse polymer sequences are useful, for example, in nucleic acid analysis, gene expression monitoring, receptor and nucleic acid binding studies, surface based DNA computation, and integrated electronic circuits and other miniature device fabrication.
Methods of synthesizing polymer sequences such as nucleotide and peptide sequences are known. Methods of synthesizing oligonucleotides are found in, for example, Oligonucleotide Synthesis: A Practical Approach, Gait, ed., IRL Press, Oxford (1984), incorporated herein by reference in its entirety for all purposes. The so-called xe2x80x9cMerrifieldxe2x80x9d solid phase peptide synthesis has been in common use for several years and is discussed in Merrifield, J. Am. Chem. Soc. (1963) 85:2149-2154, incorporated herein by reference for all purposes. Solid-phase synthesis techniques have been provided for the synthesis of several peptide sequences on, for example, a number of xe2x80x9cpins.xe2x80x9d See e.g., Geysen et al., J. Immun. Meth. (1987) 102:259-274, incorporated herein by reference for all purposes. Other solid-phase techniques involve, for example, synthesis of various peptide sequences on different cellulose disks supported in a column. See Frank and Doring, Tetrahedron (1988) 44:6031-6040, incorporated herein by reference for all purposes. Still other solid-phase techniques are discussed in U.S. Pat. No. 4,728,502 (issued to Hamill) and PCT Publication No. WO 90/00626 (Beattie, inventor).
Each of the above techniques produces only a relatively low density array of polymers. For example, the technique discussed in Geysen et al. is limited to producing 96 different polymers on pins in the dimensions of a standard microtiter plate.
Improved methods of forming high density arrays of peptides, polynucleotides, and other polymer sequences in a short period of time have been devised using combinatorial solid phase synthesis. Very Large Scale Immobilized Polymer Synthesis (VLSIPS) technology has greatly advanced combinatorial solid phase polymer synthesis and paved the way to clinical application of deoxyribonucleic acid (DNA) array chips such as those sold under the trademark GENECHIP. See Kozal et al., Nature Medicine, Vol. 2, pp. 753-759 (1996), incorporated herein by reference in its entirety for all purposes. VLSIPS technology is disclosed in Pirrung et al., U.S. Pat. No. 5,143,854 (see also PCT Publication No. WO 90/15070), Fodor et al., PCT Publication No. WO 92/10092, and PCT Publication No. WO 95/11995, Fodor et al., Science (1991) 251:767-777, all incorporated herein by reference in their entirety for all purposes. Known embodiments of VLSIPS technology employ radiation-labile protecting groups and photolithographic masks to achieve spatially defined combinatorial polymer synthesis on a substrate surface. In those embodiments, masks are used to control the selective exposure to radiation in specific locations of a surface provided with linker molecules containing radiation-labile protecting groups. In the exposed locations, the radiation-labile protecting groups are removed. The surface is then contacted with a solution containing a desired monomer. The monomer has at least one site that is reactive with the newly exposed reactive moiety on the linker and at least a second reactive site protected by one or more radiation-labile protecting groups. The desired monomer is then coupled to the unprotected linker molecules. The process can be repeated to synthesize a large number of polymer sequences in specific locations.
Other methods for synthesizing high density polymer arrays employ blocks containing channels for reagent delivery at preselected sites on the substrate. See PCT Publication No. WO 93/09668, incorporated herein by reference for all purposes. In certain embodiments, a block is contacted with the substrate and the reagents necessary to form a portion of the immobilized polymer are permitted to access the substrate via the channel(s). The block or substrate can be rotated and the process repeated to form arrays of polymers on the substrate. The block channel method can be combined with light-directed methodologies.
Certain embodiments of the present invention provide novel methods, compositions, and devices useful in synthesizing novel high density arrays of diverse polymer sequences. The polymer sequences are fashioned from individual synthesis intermediates and include diverse naturally or non-naturally occurring peptides, nucleotides, polypeptides or polynucleotides. The methods of the present invention utilize a novel chemical amplification process using a catalyst system which is initiated by radiation to assist in the synthesis the polymer sequences. Methods of the present invention include the use of photosensitive compounds which act as catalysts to chemically alter the synthesis intermediates in a manner to promote formation of polymer sequences. Such photosensitive compounds include what are generally referred to as radiation-activated catalysts (RACs), and more specifically photo activated catalysts (PACs). The RACs can by themselves chemically alter the synthesis intermediate or they can activate an autocatalytic compound which chemically alters the synthesis intermediate in a matter to allow the synthesis intermediate to chemically combine with a later added synthesis intermediate or other compound.
According to one embodiment of the present invention, one or more linker molecules are bound to or otherwise provided on the surface of a substrate, such as a glass plate. The unbound portion of the linker molecule, also referred to as the terminal or free end of the linker molecule, has a reactive functional group which is blocked, protected or otherwise made unavailable for reaction by a removable protective group. Once the protective group is removed, the functional group is made available for reaction, i.e. the reactive functional group is unblocked. A photo activated catalyst (PAC) is also located or otherwise provided on the surface of the substrate in the vicinity of the linker molecules. An autocatalytic compound may also be present on the surface of the substrate. The photo activated catalyst by itself or in combination with additional catalytic components is referred to herein as a catalyst system.
Using lithographic methods and techniques well known to those of skill in the art, a set of first selected regions on the surface of the substrate is exposed to radiation or certain wavelengths. The radiation activates the PAC which then either directly or through an autocatalytic compound catalytically removes the protecting group from the linker molecule making it available for reaction with a subsequently added synthesis intermediate.
According to one embodiment of the present invention, the radiation causes the structure of the PAC to change and to produce a catalyst capable of initiating the autocatalytic compound, also referred to herein as an enhancer, to undergo a reaction producing at least one product that removes the protective groups from the linker molecules in the first selected regions. The use of PACs and autocatalytic compounds advantageously amplifies through catalysis the number of linker molecules having their protective groups removed. Stated differently, the radiation initiates a chemical reaction which catalyzes the removal of a large number of protective groups. With the protective groups removed, the reactive functional groups of the linker molecules are made available for reaction with a subsequently added synthesis intermediate or other compound.
The substrate is then washed or otherwise contacted with an additional synthesis intermediate that reacts with the exposed functional groups on the linker molecules to form a sequence. In some preferred embodiments, the enhancers are autocatalytic compounds or groups that undergo autocatalysis when initiated by a RAC such as a PAC. The synthesis intermediate also has a reactive functional group which is blocked or otherwise made unavailable for reaction by a removable protective group. In this manner, a sequence of monomers of any desired length can be created by stepwise irradiating the surface of the substrate to initiate a catalytic reaction to remove a protective group from a reactive functional group on a already present synthesis intermediate and then introducing a monomer, i.e. a synthesis intermediate, that will react with the reactive functional group, and that will have a protective group for later removal by a subsequent irradiation of the substrate surface.
Accordingly, a second set of selected regions on the substrate which may be the same or different from the first set of selected regions on the substrate is, thereafter, exposed to radiation and the removable protective groups on the synthesis intermediates or linker molecules are removed. The substrate is then contacted with an additional subsequently added synthesis intermediate for reaction with exposed functional groups. This process is repeated to selectively apply synthesis intermediates until polymers of a desired length and desired chemical sequence are obtained. Protective groups on the last added synthesis intermediate in the polymer sequence are then optionally removed and the sequence is, thereafter, optionally capped. Side chain protective groups, if present in the polymer sequence, are also removed. The technique, when it employs photon radiation, is referred to as xe2x80x9cphotochemical amplification for the synthesis of patterned arraysxe2x80x9d or xe2x80x9cPASPAxe2x80x9d.
According to one embodiment of the present invention, the RAC produces an acid when exposed to radiation; the enhancer is an ester labile to acid catalyzed thermolytic cleavage which itself produces an acid; the protecting group is an acid removable protecting group, and the monomer is a nucleotide containing an acid removable protecting group at its C-5xe2x80x2 hydroxyl group, for example when synthesis is carried out in the 3xe2x80x2 to 5xe2x80x2 direction. It is to be understood that the teachings of the present invention are equally useful in carrying out synthesis of polynucleotides in the 5xe2x80x2 to 3xe2x80x2 direction. In that instance, the protective group is present at the 3xe2x80x2 hydroxyl group. In an alternate embodiment of the present invention, the monomer is an amino acid containing an acid removable protecting group at its amino or carboxy terminus and the linker molecule terminates in an amino or carboxy acid group bearing an acid removable protective group.
Using the techniques disclosed herein, it is possible to advantageously irradiate relatively small and precisely known locations on the surface of the substrate. The radiation does not directly cause the removal of the protective groups, such as through a photochemical reaction upon absorption of the radiation by the synthesis intermediate or linker molecule itself, but rather the radiation acts as a signal to initiate a chemical catalytic reaction which removes the protective group in an amplified manner. Therefore, the radiation intensity as used in the practice of the present invention to initiate the catalytic removal by a catalyst system of protecting groups can be much lower than, for example, direct photo removal, which can result in better resolution when compared to many non-amplified techniques.
The present invention is advantageous because it makes possible the synthesis of polymers of any desired chemical sequence at known locations on a substrate with high synthesis fidelity, small synthesis feature, and improved manufacturability. Embodiments of the present invention are useful in fabricating high density nucleic acid probe arrays or immobilizing nucleic acid sequences on a surface of a substrate. High density nucleic acid probe arrays provide an efficient means to analyze nucleic acids, to monitor gene expression and to perform computation.
It is therefore an object of the present invention to provide methods of manufacturing high density polymer arrays using chemical amplification techniques. It is a further object of the present invention to provide methods of manufacturing polymer arrays using less time and lower radiation intensities to improve polymer purity, to improve the spatial resolution and contrast between polymer and substrate and to decrease the area on the substrate where polymer sequences can be synthesized allowing many and different polymer sequences on the same substrate. It is a still further object of the present invention to provide methods of removing protecting groups from synthesis intermediates in the formation of polymer sequences using photosensitive compounds to initiate catalytic reactions. It is an even still further object of the present invention to improve precision, contrast, and ease of manufacture in the production of polymer arrays.